System and method for increasing the contrast of an image produced by an epifluorescence microscope

ABSTRACT

The contrast of an image produced by epifluorescence microscopy may be increased by placing a dichroic reflector behind the sample. The dichroic reflector reflects the emission light emitted by the fluorescent tags in the sample back through the objective lens while allowing the shorter wavelength excitation light to pass through the sample holder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application of continuation application U.S. patentapplication Ser. No. 11/225,460, filed Sep. 13, 2005, and U.S. patentapplication Ser. No. 10/102,500, filed Mar. 19, 2002, which claimspriority of U.S. Provisional Application No. 60/276,906, filed Mar. 19,2001; the entire contents of which are incorporated herein by referencein their entirety.

FIELD OF THE INVENTION

The present invention relates to microscope slides and the like for usein epifluorescence microscopy of biological specimens.

BACKGROUND OF THE INVENTION

Citation or identification of any reference in this section or anysection of this application shall not be construed as an admission thatsuch reference is available as prior art to the present invention.

An epifluorescence microscope is similar to a conventional reflectingoptical microscope in that both microscopes illuminate the sample andproduce a magnified image of the sample. An epifluorescence microscope,however, uses the emitted fluorescent light to form an image whereas aconventional reflecting optical microscope uses the scatteredillumination light to form an image. The epifluorescent microscope usesa higher intensity illumination, or excitation, light than aconventional microscope. The higher intensity excitation light is neededto excite a fluorescent molecule in the sample thereby causing thefluorescent molecule to emit fluorescent light. The excitation light hasa higher energy, or shorter wavelength, than the emitted light. Theepifluorescence microscope uses the emitted light to produce a magnifiedimage of the sample. The advantage of a epifluorescence microscope isthat the sample may be prepared such that the fluorescent molecules arepreferentially attached to the biological structures of interest therebyproducing an image of the biological structures of interest.

A common problem in epifluorescence microscopy is the low contrast, orlow signal-to-noise (S/N) ratio, of the fluorescent image. This is dueto the low intensity of the emitted light compared to the high intensityof the excitation light. A dichroic mirror is usually used to reduce thescattered excitation light before the image is viewed or recorded.

The dichroic mirror is only partially effective in removing theexcitation light from the emitted light so other measures must be takento increase the S/N ratio of the fluorescent image. In order to assistin the discussion of the other approaches to increasing the S/N ratio ofthe fluorescent image, reference to FIG. 1 is helpful.

FIG. I illustrates the optical path and components of a typicalepifluorescence microscope. A sample 100 is placed on a sample holder105, which is normally a microscope slide. The sample is prepared priorto being placed on the holder 105 with fluorescent tags that bind to thebiological structures of interest. The fluorescent tags may be a singletype of fluorescent tag that binds to a particular biological structureor may be a mixture of several fluorescent tag types with each tag typebinding to a different biological structure. The sample 100 isilluminated by a light source 110 that produces the excitation lightwith sufficient intensity to cause the tags to emit fluorescent light.The excitation light generated by the light source 110 follows a path115 through an excitation filter 120 that acts as a band-pass filterallowing only a narrow range of frequencies to pass through theexcitation filter 120. The excitation filter 120 is chosen to allow onlythe light of a frequency that will cause the tags to fluoresce. Theexcitation light is reflected by a dichroic mirror 130 into theobjective lens 140 of the microscope following path 125. A dichroicmirror separates the excitation light from the emission light, in thisexample, by reflecting the excitation light while transmitting theemission light. The excitation light propagates through the objectivelens 140 and illuminates the sample 100 and excites the tags in thesample to emit fluorescent light, also referred to as emission light.The emission light propagates along path 125 in the opposite directionas the excitation light. The emission light passes through the objectivelens 140 and through the dichroic mirror 130 and continues along path135 through an emission filter 150. The emission filter 150 is selectedto allow only light matching the frequency of the emission light to passthrough the filter. The emission filter 150 may be a band-pass fitter,or a long-pass filter that allows the longer wavelength emission lightto pass through while stopping the shorter wavelength excitation light.After filtering by the emission filter 150, the emission light is formedinto an image by an imaging lens 160. If the emission filter 150 isperfectly efficient in removing all but the emission light, themagnified fluorescent image would have a very high contrast and S/Nratio. Unfortunately, emission filters are not perfectly efficient so asmall amount of excitation light is transmitted though the emissionfilters. Because the intensity of the excitation light is very high, thesmall fraction of excitation light that passes through the emissionfilter is sufficient to severely degrade the contrast of the fluorescentimage. In addition, the excitation frequency is usually very close tothe emission frequency of the fluorescent tag molecule. The closeness ofthe two frequencies adds a further requirement on the emission filterthat the filter have a very steep adsorption edge between the emissionfrequency and excitation frequency.

U.S. Pat. No. 6,094,274 issued on Jul. 25, 2000 to Yokoi teaches the useof two interference films as an emission filter. The two interferencefilms act to sharpen the adsorption edge between the emission frequencyand excitation frequency. The sharp adsorption edge blocks more of theexcitation light while transmitting more of the emission light to theimaging lens.

Another approach to increasing the S/N ratio of a fluorescent image isdisclosed in Japanese Application Publication No. 9-292572 by Sudo, etal. published on Nov. 11, 1997 (hereinafter referred to as “Sudo”). Sudodiscloses the use of a mirror behind the sample that reflects theexcitation light back through the sample. The reflected excitation lightapproximately doubles the excitation light seen by the sample andtherefore approximately doubles the amount of emission light given offby the sample. A portion of the reflected excitation light will,however, also pass through the dichroic mirror and emission filteradding to the “noise” of the higher emission signal. In addition, theincreased illumination of the sample from the reflected excitation lightincreases the bleaching effect on the tagged sample. Bleaching occurswhen the fluorescent tag molecules emit decreasing amounts offluorescent light as the molecules are illuminated by the excitationlight. For example, a fluorescent tag molecule will emit less than 10%of its emission intensity after only a minute of being illuminated bythe excitation light. As the intensity of the excitation light increasesthe bleaching rate increases thereby decreasing the emission light andreducing the contrast of the fluorescent image.

Therefore, there still remains a need to provide a microscope systemcapable of producing a high contrast fluorescent image while reducingunnecessary bleaching of the sample.

SUMMARY OF THE INVENTION

One aspect of the present invention is directed to an epifluorescencemicroscope for imaging a biological sample having fluorescent tagmolecules, the tag molecules emitting an emission light at an emissionfrequency when illuminated by an excitation light having an excitationfrequency, the microscope comprising: an excitation light sourcegenerating an excitation light; a first dichroic mirror reflecting theexcitation light; an objective lens disposed to receive the excitationlight reflected by the dichroic mirror and to illuminate the sample withthe excitation light; an imaging lens disposed to receive emission lightfrom the sample through the objective lens and first dichroic mirror;and a dichroic sample reflector disposed behind the sample reflectingthe emission light back through the sample, objective lens, firstdichroic mirror and imaging lens, while transmitting the excitationlight through the reflector.

Another aspect of the present invention is directed to a sample holderfor supporting a sample for epifluorescence microscopy, the sampleemitting an emission light when illuminated by an excitation light, thesample holder comprising a base and a dichroic reflector disposed on thebase, wherein the dichroic reflector reflects the emission light emittedby the sample while transmitting the excitation light illuminating thesample.

Another aspect of the present invention is directed to a microscopeslide for supporting a sample, the slide comprising a top surface and aninfra-red reflecting film deposited on the top surface, the filmdirectly supporting the sample.

Another aspect of the present invention is directed to a sample bolderholding a sample for an epifluorescence microscope, the sample emittingan emission light when illuminated by an excitation light, the sampleholder comprising: a base supporting the sample; and a sample reflectordisposed on the base between the sample and base, wherein the reflectorreflects the emission light emitted by the sample while transmitting theexcitation light illuminating the sample, wherein the sample reflectoris concave having a focal point disposed in the sample. Another aspectof the present invention is directed to a sample holder for supporting asample emitting an emission light when illuminated by an excitationlight, the sample holder comprising: a top surface for directlysupporting a sample, the top surface having an infra-red reflecting filmdeposited on the top surface; and a bottom surface having a dichroicfilm deposited on the bottom surface, the dichroic film reflectingemission light and transmitting excitation light.

Another aspect of the present invention is directed to a sample holderfor supporting a sample emitting an emission light when illuminated byan excitation light, the sample holder comprising: a top surface fordirectly supporting a sample; and a dichroic film deposited on the topsurface, the dichroic film transmitting excitation light and reflectingemission light.

Another aspect of the present invention is directed to a method forincreasing the contrast of an image produced by an epifluorescencemicroscope of a sample emitting an emission light when illuminated by anexcitation light comprising the steps of: illuminating the sample withthe excitation light; collecting a first portion of the emission light;reflecting a second portion of the emission light; collecting thereflected portion of the emission light; and producing an image usingthe collected first portion of the emission light and the collectedreflected portion of the emission light.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention may be understood more fully by reference to thefollowing detailed description of the preferred embodiments of thepresent invention, illustrative examples of specific embodiments of theinvention and the appended figures in which:

FIG. 1 is a view of a conventional epifluorescence microscope.

FIG. 2 is a view of an embodiment of the present invention.

FIG. 3 is a detail view of the sample holder of the embodiment shown inFIG. 2.

FIG. 4 is a view of another embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIG. 2 is a view of an embodiment of the present invention. Excitationlight generated by a light source 210 is filtered by an excitationfilter 220. The excitation filter 220 is preferably a band-pass filterallowing excitation frequencies matched to the fluorescent tags in thesample to pass through while absorbing the rest. The excitation light isredirected by reflection from a dichroic mirror 230 through an objectivelens 240 to illuminate a sample 200 having fluorescent tag molecules.The excitation light causes the fluorescent tag molecules to emitfluorescent light. The fluorescent light emitted by the tag molecules iscollected by the objective lens 240 and is transmitted through thedichroic mirror 230. The dichroic mirror 230 is selected to reflect theexcitation light emitted by the light source 210 toward the sample 200while transmitting the emission light emitted by the sample through thedichroic mirror. The emission light is filtered by an emission filter250 to remove extraneous light such as scattered excitation light. Theemission light is formed into an image by an imaging lens 260. Thedetails of mounting and aligning the optical elements described aboveare known to one of ordinary skill in the optical microscopy art and aretherefore not discussed.

The sample 200 is supported by a sample holder 205. The sample holder205 includes a sample reflector 207 positioned directly behind thesample 200. It is understood that the term “behind” is relative to thedirection of the incident excitation light. The sample reflector 207, ina preferred embodiment, is a dichroic mirror selected to reflect theemission light while transmitting the excitation light.

FIG. 3 is a detail view of the sample holder 205 and sample reflector207. A sample 300 such as a blood or cell smear is placed on a samplesupport 305 such as a glass slide. The sample is treated with afluorescent tag that preferentially adsorbs to the biological structuresof interest. The sample 300 and sample support 305 are supported by asample holder 310. The sample holder 310 has a base 315 supporting areflector 320 that, in turn, supports the glass slide 305 and sample300.

Excitation light 350 illuminates the sample 300 and interacts with thesample 300, sample support 305 and reflector 320. For example, theexcitation light 350 may be back-scattered from the sample, shown as ray352, or may be back-scattered from the sample support 305, shown as ray354, or may be backscattered from the reflector, shown as ray 356. Someof the back-scattered light 352 354 356 is collected by the objectivelens (not shown) and transmitted through to the imaging lens. Theback-scattered light 352 354 356 collected by the imaging lenscontributes to the background noise level of the image and thereforereduces the S/N ratio of the image.

A small fraction of the excitation light 350 interacts with thefluorescent tags 302 causing the fluorescent tags 302 to emitfluorescent light 360 362. Some of the emission light 360 is collectedby the objective lens and imaged by the imaging lens thereby forming theimage of the biological structures of interest. Less than one-half ofthe emission light 360 362 is directly collected by the objective lensbecause at least one-half of the emission light is emitted in adirection away from the objective lens as represented by ray 362.

In a preferred embodiment, the reflector 320 is a dichroic mirror thatallows the short wave-length excitation light 351 to pass through themirror 320 while reflecting the longer wave-length emission light 362.Selection of the reflector 320 to match the excitation and emissionfrequencies of the specific fluorescent tag molecule used to prepare thesample is well known to one of ordinary skill in the fluorescentmicroscopy art.

The novel feature of the reflector 320 is that, unlike the dichroicmirror commonly used in typical epifluorescent microscopes, thereflector 320 reflects the emission light instead of the excitationlight. In the preferred embodiment, the reflector 320 transmits orabsorbs most of the excitation light 350 and therefore reduces theamount of back-scattered excitation light 356 that may be collected bythe objective lens. Reducing the amount of back-scattered excitationlight 356 also reduces the noise in the image and results in a highercontrast image of the sample. In addition, the reflected emission light362 may be collected by the objective lens and contribute to the “signalportion” of the image and thereby create a higher contrast image.

The reflected emission light 362 is reflected from the surface of thereflector 320. The reflector surface is behind, with respect to thedirection of the excitation light, the tag molecule in the sample andtherefore will not be in the same focal plane 370 as the sample. Theresulting image will have a higher intensity due to the reflectedemission light but will have a lower resolution due to the spatialdisplacement of the reflector surface with respect to the plane of thesample. In many situations, the higher intensity image is more importantthan the slight loss of resolution. For example, if the emission lightis used to detect the presence of a rare cell in a sample, a brighterimage is preferred because a bright image is easier to detect. Theslight loss in resolution in this example is not as important becausethe detection of the rare cell depends primarily on image brightness,not image resolution.

In another embodiment of the present invention, the sample is placeddirectly on the reflector 320. Placing the sample directly on thereflector 320 eliminates the need for a sample support 305 and reducesthe distance between the plane of the reflector and the plane of thesample 370 thereby reducing the focal mismatch between the image formedby the emission light collected directly from the sample and the imageformed by the reflected emission light 362.

The reflection surface may also be used as a reference plane forautomatically focusing the image using laser tracking such as theTeletrac LTAF8000 series Laser Tracking Autofocus from AxsysTechnologies of Rocky Hill, Conn. In typical auto-focusing methods, theimage is focused based on the reflected light from a surface, usually acover slide. In typical laser autofocusing systems, the frequency of thelaser light is usually in the infrared portion of the spectrum and has alonger wavelength than the light emitted by the fluorescent tags. Theamount of reflected light is usually less than about 5% of the incidentlight. The small signal strength of the reflected light causes themicroscope to lose focus if the sample is perturbed. In an embodimentwhere the reflector acts as a high-pass filter allowing the higherfrequency excitation light through the filter while reflecting the lowerfrequency emission and autofocusing light back through the objectivelens. Although the reflector may not reflect all of the infra-redfocusing light, a sufficient amount of focusing light will be reflectedfor the laser auto-focus system to maintain focus on the top surface ofthe reflector.

FIG. 4 is a side view of another embodiment of the present invention. Aninfra-red reflecting film 410 is deposited on the top surface of asample support 420 and a dichroic film 430 reflecting emission lightwhile transmitting excitation light is deposited on the bottom surfaceof the sample support. The sample support may be a single-use disposableglass slide. The sample 405 is placed directly on the infra-redreflector 410 and illuminated by both the excitation light 450 and afocusing beam 460. The focusing beam 460 is preferably an infra-redbeam, characterized by a wavelength between 700-800 nm, that is part ofa laser auto-focus system such as the one described above. The focusingbeam 460 is reflected (indicated by ray 465) by the infra-red reflectingfilm 410 back to the laser auto-focus system that automatically focusesthe microscope on the infra-red reflecting film 410. In most situations,the biological structures of interest usually settle onto the surface ofthe infra-red reflecting film 410. Therefore, focusing on the reflectingfilm 410 will likely bring the biological structures of interest intofocus. The dichroic film 430 on the bottom surface of the sample support420 will reflect the emission light (indicated by ray 455) back throughthe sample for collection by the objective lens while transmitting orabsorbing the excitation light (indicated by ray 451).

In a preferred embodiment, the infra-red reflecting film 410 is metalfilm, such as for example titanium, between 0.6-90 nm. The metal filmmay be deposited using any of the known techniques for depositing thinfilms such as physical deposition. In a preferred embodiment, magnetronsputtering may be used to apply the infra-red reflecting film to theglass slide. The sputtering composition, in a preferred embodiment, issubstantially titanium with impurities such as carbon, nitrogen, iron,oxygen, and hydrogen cumulatively comprising less than 1% of thesputtering composition. Other sputtering compositions comprising metalsdifferent than titanium may be used to form the metal film.

The selection of the sputtering composition and film thickness maydetermined by one of skill in the art by measuring the intensity of thereflected auto-focus beam from the reflecting film. In one embodiment ofthe present invention, the thickness and composition of the film isadjusted to reflect between 4-8% of the incident infra-red auto-focusbeam. In a preferred embodiment, the thickness and composition of thefilm is adjusted to reflect between 5.5-7% of the incident auto-focusbeam.

In other situations, however, a high contrast, high resolution image ispreferred. In another embodiment of the present invention, the reflectoris shaped into a concave surface having a focal point in the plane(defined by the excitation light ray) of the sample. This has theadvantage of being able to focus both the direct and reflected emissionlight on the same focal plane.

In another embodiment of the present invention, more than one kind offluorescent tag may be used to image different biological structures ofthe sample.

A mixture of different kinds of fluorescent tag molecules is used toprepare the sample. Each kind of fluorescent tag attaches to differentbiological structures. The light emitted by the fluorescent tags mayhave a different frequency and the excitation light required to causethe tags to fluoresce may have a different frequency depending on thekind of fluorescent tag. Each tag may require its own set of excitationand emission filters selected for the excitation and emission lightfrequencies of the specific tag. The sample holder reflector is chosento transmit or absorb all the excitation frequencies of the fluorescenttags while reflecting all the emission frequencies of the fluorescenttags.

The invention described herein is not to be limited in scope by thepreferred embodiments herein described, since these embodiments areintended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. For example, instead oftransmitting the excitation light, the sample reflector may absorb theexcitation light. Another example includes the use of a laser as theexcitation light source. Since the laser produces essentiallymonochromatic light, using a laser as the excitation light sourceeliminates the need for an excitation filter. Such modifications arealso intended to fall within the scope of the invention.

1. An epifluorescence microscope for imaging a biological sample havingfluorescent tag molecules, the tag molecules emitting an emission lightat an emission frequency when illuminated by an excitation light havingan excitation frequency, the microscope comprising: an excitation lightsource generating an excitation light; a first dichroic mirrorreflecting the excitation light; an objective lens disposed to receivethe excitation light reflected by the dichroic mirror and to illuminatethe sample with the excitation light; an imaging lens disposed toreceive emission light from the sample through the objective lens andfirst dichroic mirror; and a dichroic sample reflector disposed behindthe sample reflecting the emission light back through the sample,objective lens, first dichroic mirror and imaging lens, whiletransmitting the excitation light through the reflector.
 2. Theepifluorescence microscope of claim 1 wherein the dichroic samplereflector is concave having a focal point disposed in the sample.
 3. Theepifluorescence microscope of claim 1 wherein said biological sample isplaced directly on the dichroic sample reflector.
 4. A method forincreasing the contrast of an image produced by an epifluorescencemicroscope of a sample emitting an emission light when illuminated by anexcitation light comprising the steps of: illuminating the sample withthe excitation light; collecting a first portion of the emission light;reflecting a second portion of the emission light; collecting thereflected portion of the emission light; and producing an image usingthe collected first portion of the emission light and the collectedreflected portion of the emission light.
 5. The method in claim 4wherein the reflected portion of the emission light is collected from afocal point disposed in said sample.
 6. The method of claim 4 whereinsaid sample is placed directly on a dichroic sample reflector.